All animals have a number of molecular and cellular components capable of interacting with and neutralizing various harmful foreign substances (antigens) in their environment. An animal's immune response to antigen involves both non-specific molecules and cells, as well as systems and mechanisms for the development of protective responses which possess memory and are extremely specific.
The primary cells of the immune system are the white blood cells, called lymphocytes, which are derived from cells in the bone marrow. One class of lymphocytes, the T lymphocytes, mature under the influence of the thymus and, upon stimulation by antigen, give rise to cellular immunity. T lymphocytes are also involved in the regulation of B lymphocytes, which, upon appropriate stimulation, mature into plasma cells that secrete antibody.
Mature T lymphocytes that emerge from the adult mammalian thymus migrate to peripheral lymphoid organs such as the spleen and lymph nodes. There, the naive T cells encounter antigens, usually in the form of processed peptides, bound to self molecules encoded by the major histocompatibility complex.
MHC class II molecules display peptides derived from proteins internalized through the endocytic pathway and are recognized predominantly by inducer T lymphocytes expressing the CD4 surface molecule. MHC class I molecules display peptides derived from proteins synthesized inside the antigen-presenting cell (for example, viral proteins) and are largely recognized by cytotoxic T lymphocytes expressing the CD8 surface molecule. Germain, Nature, 322:687 (1986).
The frequency of T cells specific for any given foreign antigen is initially small. If these cells are to play a central role in host defense, they must selectively increase in number. Thus, activation of the T lymphocyte upon recognition of foreign antigen leads to autocrine growth in which the stimulated naive cells proliferate in response to their own production of the polypeptide growth hormone interleukin-2 (IL-2) and the receptor for IL-2. Smith, Annu. Rev. Immunol., 2:319 (1984); Greene et al, Annu. Rev. Immunol., 4:69 (1986); Waldman, Annu. Rev. Biochem., 58:875 (1989). In addition, the cells differentiate, acquiring the ability to produce other lymphokines, such as interleukin-4 (IL-4) and gamma interferon (IFN-.gamma.) for CD4.sup.+ cells. Swain et al, J. Immunol., 141:3445 (1988); Salmon et al, J. Immunol., 143:907 (1989); Gajewski et al, Immunol. Rev., 111:79 (1989). These proteins serve as effector molecules for activating other cells in the immune system. IL-2 also plays a critical role in this recruitment function, as it can act in a paracrine fashion to help activated B lymphocytes and CD8.sup.+ cytotoxic T lymphocytes expand in number.
The minimal requirement for an antigen-specific immune response is the effective binding of the processed peptide and the MHC molecule on an antigen-presenting cell by a clonally distributed T cell receptor for antigen. For most T cells, the T cell antigen receptor is a heterodimeric glycoprotein composed of two glycosylated protein chains, one of which is designated the alpha and the other, the beta, chain. Each of the two proteins chains is divided into variable (V) and constant (C) regions. The variable portions of the protein chains differ between T cell clones and are primarily responsible for the unique recognition specificity of a given T cell. These chains are non-covalently associated with another cell surface molecule, designated CD3, which is believed to be involved in signal transduction.
Although occupancy of the T cell receptor complex (TCR) by antigen in association with the major histocompatibility complex (MHC) is necessary for the initiation of T cell activation, several lines of evidence suggest that a second costimulatory signal is essential for the induction of proliferation and lymphokine secretion, particularly of interleukin-2. Schwartz, Science, 248:1349 (1990); Kawakami et al, J. Immunol., 142:1818 (1989); Mueller et al, J. Immunol., 142:2617 (1989); Williams et al, J. Immunol., 145:85 (1990). In murine and human systems, one type of costimulatory signal is delivered by antigen presenting cells (APC) and requires cell to cell contact. Kawakami et al, J. Immunol., 142:1818 (1989); Williams et al, J. Immunol., 145:85 (1990). Cells which can deliver this costimulatory signal include activated, but not resting B lymphocytes (Ashwell et al, J. Exp. Med., 159:881 (1984)); gamma-interferon (.gamma.-INF) activated monocytes, and dendritic cells (Kawakami et al, J. Immunol., 142:1818 (1989); Matis et al, Proc. Natl. Acad. Sci. USA, 80:6019 (1983)).
Several recent studies in human systems have provided compelling evidence that the B cell activation antigen B7 can provide one such costimulatory signal. Gimmi et al, "B7 provides a costimulatory signal which induces T cells to proliferate and secrete interleukin-2", Proc. Natl. Acad. Sci. USA, (in press); Linsley et al, J. Exp. Med., 173:721 (March 1991); Koulova et al, J. Exp. Med., 173:759 (March 1991).
The B7 activation antigen is a cell surface molecule that appears on the surface of a subpopulation of B lymphocytes within 24 hours after activation with EBV or anti-immunoglobulin. Freedman et al, J. Immunol., 139:3260-3267 (1987). This antigen is present on a subpopulation of human splenic B lymphocytes that respond more rapidly to signals of B cell activation and proliferation. Specifically, B7+ B cells are not capable of independently responding to low molecular weight B cell growth factor or IL-2. However, after activation, the B7+ subpopulation of B cells more rapidly enters the S phase of the cell cycle in response to growth factors. The B7 antigen thus identifies a subpopulation of B cells that appear to be previously activated or primed in vivo and demonstrate accelerated growth to subsequent triggers.
Within the hematopoietic system, B7 is expressed on activated B cells and on monocytes that have been activated with gamma-interferon. In addition, B7 is present on some B lymphoblastoid and neoplastic cell lines, and on some tumor cells isolated from patients with certain types of B cell malignancies, particularly lymphomas.
B7 has recently been shown to be an adhesion ligand for another member of the immunoglobulin superfamily, the T cell surface protein CD28. Freeman et al, J. Immunol., 143:2714 (1989); Aruffo et al, Proc. Natl. Acad. Sci. USA, 84:8573 (1987); Linsley et al, Proc. Natl. Acad. Sci. USA, 87:5031 (1990); Williams et al, Ann. Rev. Immunol., 6:381 (1988). CD28 is constitutively expressed on 95% of human CD4.sup.+ T cells, 50% of CD8.sup.+ T cells, and on thymocytes which co-express CD4 and CD8. Turka et al, J. Immunol., 144:1646 (1990); Yamada et al, Eur. J. Immunol., 15:1164 (1985); Martin et al, J. Immunol., 136:3282 (1986). Following suboptimal activation of T cells with anti-CD3 mAb; (Martin et al, J. Immunol., 136:3282 (1986)); anti-CD2 mAb, or phorbol ester; (June et al, J. Immunol., 143:153 (1989)) crosslinking of CD28 by anti-CD28 mAb results in enhanced T cell proliferation and greatly augments synthesis of multiple lymphokines. Thompson et al, Proc. Natl. Acad. Sci. USA, 86:1333 (1989). A method of immunotherapy involving stimulation of the T cell CD28 surface molecule to enhance T cell proliferation and increase lymphokine levels involving anti-CD28 monoclonal antibodies has been described. PCT International Publication Number WO 90/05541.
That B7 is likely to be an important regulator of T cell proliferation and lymphokine production is evidenced by its pattern of expression and functional activity described above. Further, human B7 transfected cells or recombinant B7-Ig fusion protein augment proliferation and induce interleukin-2 (IL-2), but not interleukin-4 (IL-4), synthesis in T cells which have been treated with phorbol ester or anti-CD3 mAb. Gimmi et al, "B7 provides a costimulatory signal which induces T cells to proliferate and secrete interleukin-2", Proc. Natl. Acad. Sci. USA, (in press); Linsley et al, J. Exp. Med., 173:721 (1991); Koulova et al, J. Exp. Med., 173:759 (1991).
Approaches to either upregulate or block the expression of B7 or the ligation of B7 to its natural ligand on T cells would provide a specific means of therapeutic intervention, to respectively enhance or suppress T cell-mediated immune responses in vivo. One approach involves the molecular cloning of B7, which would enable the recombinant preparation of B7 proteins. However, although the molecular structure of a number of other human B cell activation antigens has previously been determined, prior to the present invention, attempts to clone B7 were unsuccessful. Previously cloned B cell associated or restricted activation antigens include the nonlineage-restricted activation antigen 4F2 and transferrin receptor as well as the lymphoid-associated activation antigens, intracellular adhesion molecule-1, CD25, Blast-1 and CD23. The cDNA clones encoding various human B-cell associated antigens have been characterized through the use of expression techniques described by Aruffo and Seed (Proc. Natl. Acad. Sci., 84:8573-8577 (1987); Proc. Natl. Acad. Sci., 84:3365-3369 (1987)), including those encoding the B cell associated antigens CD19, CD20, CD22, CD27, CD39 and CDw40.
It is an object of the present invention to molecularly clone genes encoding the B7 activation antigen.
Another object of the invention is to provide nucleic acid molecules which code for the human and murine B7 B lymphocyte activation antigen.
Yet another object of the present invention is to provide a diagnostic method for quantitatively measuring activated B-cells in a biological sample.
A still further object of the present invention is to provide recombinantly produced B7 proteins.
These as well as other objects and advantages will be apparent from the following specification, drawing and claims.